The present application is the national stage under 35 U.S.C. 371 of PCT/IL99/00709, filed Dec. 30, 1999.
The present invention is directed to a method and pharmaceutical compositions for inhibiting activity of tumor necrosis factor (TNF).
Tumor necrosis factor (TNF) is a pro-inflammatory cytokine produced by a wide spectrum of cells. It has a key role in defending the host, mediating complex cellular responses of different, and even contrasting, nature (Aggarwal et al, 1996). In excess, TNF may have detrimental systemic effects. Two specific high affinity cell surface receptors, the p55 TNF-receptor (p55 TNF-R) and the p75 TNF-receptor (p75 TNF-R), function as transducing elements, providing the intracellular signal for cell responses to TNF. The extracellular parts of the TNF-Rs, known as soluble TNF-Rs, were formerly referred to as TBP-I and TBP-II respectively (see Wallach, U.S. Pat. No. 5,359,037 and Tartaglia et al., 1992; Loetscher et al., 1991).
The biological effects of TNF depend upon its concentration and site of production. At low concentrations. TNF may produce desirable homeostatic and defense functions. For example, these effects may destroy tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against infectious agents and to recovery from injury. However, at higher concentrations, systemically or in certain tissues, TNF can synergize with other cytokines, notably interleukin-1, to aggravate many inflammatory responses. Additionally, the effects of TNF-xcex1, primarily on the vasculature, are now known to be a major cause for symptoms of septic shock (Tracey et al, 1986). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia.
TNF has been found to induce the following activities (together with interleukin-2): fever, slow-wave sleep, hemodynamic shock, increased production of acute phase protein, decreased production of albumin, activation of vascular endothelial cells, increased expression of major histocompatibility complex molecules, decreased lipoprotein lipase, decreased cytochrome P450, decreased plasma zinc and iron, fibroblast proliferation, increased synovial cell collagenase, increased cyclo-oxygenase activity, activation of T cells and B cells, and induction of secretion of the cytokines, TNF itself, interleukin-1 and interleukin-6.
Because of its pleiotropic effects, TNF has been implicated in a variety of pathologic states in many different organs of the body. In blood vessels, TNF promotes hemorrhagic shock, down-regulates endothelial cell thrombomodulin, and enhances a procoagulant activity. It causes adhesion of white blood cells, and probably of platelets, to the walls of blood vessels, and so may promote processes leading to atherosclerosis, as well as to vasculitis.
TNF activates blood cells and causes the adhesion of neutrophils, eosinophils, monocytes/macrophages and T and B lymphocytes. By inducing interleukin-6 and interleukin-8, TNF augments the chemotaxis of inflammatory cells and their penetration into tissues. Thus, TNF has a role in the tissue damage of autoimmune disease, allergies and graft rejection.
TNF has also been called cachectin because it modulates the metabolic activities of adipocytes and contributes to the wasting and cachexia accompanying cancer, chronic infections, chronic heart failure and chronic inflammation. TNF may also have a role in tissue damage of autoimmune diseases, allergies, and graft rejection.
TNF also has metabolic effects on skeletal and cardiac muscle. It also has marked effects on the liver: it depresses albumin and cytochrome P450 metabolism and increases production of fibrinogen, xcex1-Acid Glycoprotein (AGP) and other acute phase proteins. It can also cause necrosis of the bowel.
In the central nervous system, TNF crosses the blood-brain barrier and induces fever, increased sleep and anorexia. Increased TNF concentration is also associated with multiple sclerosis. It also causes adrenal hemorrhage and affects production of steroid hormones, enhances collagenase and PGE-2 in the skin, and causes the breakdown of bone and cartilage by activating osteoclasts.
Thus, TNF is involved in the pathogenesis of many undesirable inflammatory conditions, in autoimmune disease, graft, rejection, vasculitis and atherosclerosis. It appears to have a role in heart failure, in the response to cancer and in anorexia nervosa. For these reasons, means have been sought to inhibit the activity of TNF as a way to control a variety of diseases.
While exploring ways for antagonizing the destructive potential of TNF in certain clinical conditions, investigators looked for natural TNF inhibitors (Engelmann et al, 1989; Engelmann et al, 1990; Seckinger et al, 1989; Olsson et al, 1989). Such agents, first detected in urine, were structurally identical to the extracellular cytokine binding domains of the two membrane associated TNF-Rs (Nophar et al, 1990). These shed soluble TNF-Rs (sTNF-Rs) can compete for TNF with the cell surface receptors and thus block the cytokine activity.
However, interactions between the TNF-Rs and their ligand are much more complex than initially thought. At physiological concentrations, the trimeric and bioactive TNF molecules decay, dissociating into inactive monomeric forms (Petersen et al, 1989; Aderka et al, 1992). Addition of sTNF-Rs to the TNF trimers promotes formation of complexes between them, which can preserve and prevent the decay of the active, trimeric forms of TNF (Aderka et al, 1991; De Groote et al, 1993). This bioactive TNF may dissociate from this complex to replace free TNF which decayed, thus maintaining a constant concentration of free, bioactive, trimeric cytokine. This reversible interaction between the soluble receptors and their ligand expands the functions attributable to the TNF receptors. In their soluble form, the TNF-Rs may serve as:
(a) TNF antagonists (when present in large excess relative to TNF);
(b) TNF carrier proteins (between body compartments);
(c) slow release reservoirs for bioactive TNF;
(d) stabilizers of the TNF""s bioactive form (which may also prolong the half-life of TNF); and
(e) TNF xe2x80x9cbuffersxe2x80x9d, by inhibiting the effects of high TNF concentrations and presenting it at low and well-controlled levels to the cells (Aderka et al, 1992).
The functions of the TNF receptors, thus, are not limited to signal transduction but include, in their soluble forms, extracellular regulatory roles affecting local and systemic bioactive TNF availability.
Examination of patients with septic shock due to meningococcemia revealed that the ratio of TNF/sTNF-Rs was higher in patients with a fatal outcome compared to patients who recovered, suggesting a critical imbalance between the ligand and its inhibitors (Girardin et al, 1994). Neutralization of the excess TNF seemed to be the preferred next step.
Indeed, dimeric Fc fusion constructs of the p55 sTNF-R, but not of the p75 sTNF-R, were found to protect mice from lethal doses of LPS (Evans et al, 1994) if administered not later than 1-3 hours post LPS (Peppel et al, 1991; Mohler et al, 1993; Ashkenazi et al, 1991; Lesslauer et al, 1991). This suggests that septic shock manifestations occur if the initial high TNF concentrations generated are not buffered by adequate soluble receptor concentrations during that narrow window of time.
To add to the growing confusion, neutralization of TNF with monoclonal anti-TNF Ab (Abraham et al, 1995; Kaul, et al, 1996) or p55 sTNF-R IgG1 (Leighton et al, 1996) in patients with severe sepsis or septic shock yielded conflicting results. In one study, the antibodies proved ineffective (Abraham et al, 1995), while in the other, administration of the antibodies benefited only those patients with baseline interleukin-6 levels higher than 1000 pg/ml but increased the mortality of those with lower interleukin-6 levels (Kaul et al, 1995). In another randomized trial, septic patients given a recombinant dimer consisting of sTNF-R/Fc portion of IgG1 had higher mortality (48-53%) as compared to placebo-treated patients (30%) (Suffredini et al, 1994; Fisher et al, 1996). Interestingly, the higher the dose of the sTNF-Rs administered, the higher was the patient mortality (Fisher et al, 1996). It was suspected that the effective removal of circulating TNF may result in the exacerbation of the systemic infection (Fisher et al, 1996). In contrast, in a recent study the administration of similar soluble Fc receptor constructs apparently benefitted septic patients irrespective of their serum interleukin-6 concentrations, with a 36% mortality reduction compared to placebo treated individuals (Leighton et al, 1996). These contradictory data give the impression that the administration of sTNF-Rs may have a very narrow therapeutic index which would be difficult to individualize at bedside. Too much of the receptors may totally neutralize TNF, exacerbating the systemic infection, while too little of the receptors may not neutralize enough TNF, resulting in septic shock and the patient""s demise. The real challenge is to fine-tune the sTNF-R dose in order to permit low TNF levels to exert their protective effects. Thus, paradoxically, lower doses of sTNF-Rs than previously employed (Fisher et al, 1996), rather than higher ones, may benefit septic shock patients.
Since the TNF neutralization should not be complete, but should be aimed to leave low amounts of bioactive TNF to exert the desired beneficial effects, natural soluble TNF receptors may be ideally suited for this purpose.
TNF is also a pivotal cytokine in the pathogenesis of Crohn""s Disease, a chronic and disabling disorder of the bowel, and is, therefore, a prime target for specific immunotherapy (Braegger et al, 1992; MacDonald et al, 1990; Breese et al, 1994). Indeed, treatment of Crohn""s Disease patients with chimeric anti-TNF monoclonal antibodies induced a spectacular remission in patients unresponsive to conventional therapy (van Dullemen et al, 1995). Whether slow release preparations of sTNF-Rs (Eliaz et al, 1966) will have identical effects on the course of this disease remains to be determined.
In another autoimmune disorder, rheumatoid arthritis, it was demonstrated that the serum sTNF-Rs may be useful in monitoring disease activity (Cope et al, 1992; Roux-Lombard et al, 1993). It was shown that despite the presence of high levels of TNF inhibitors in joints affected by rheumatoid arthritis, these inhibitors were insufficient to neutralize TNF activity (Cope et al, 1992). A randomized double blind study comparing administration of chimeric anti-TNF monoclonal antibodies to patients with rheumatoid arthritis resulted in an impressive clinical remission (Levine et al, 1994). Recently, it was demonstrated that incorporation of the sTNF-Rs into polymeric systems, such as ethylene-vinyl acetate copolymers or polylactic-glycolic acid and their subcutaneous injection, can provide systemic natural p55 sTNF-Rs at high concentrations, at a constant rate for prolonged periods (more than one month) (Eliaz et al, 1966). It is thus possible that sTNF-Rs will prove therapeutically effective in treating rheumatoid arthritis as well.
Elevated concentrations of TNF and its soluble receptors have been detected in sera of patients with heart failure (Levine et al, 1990). TNF may contribute to the impaired myocardial contraction in this condition as it was shown to produce a significant depression of myocyte shortening (Cunnion, 1990). Furthermore, whole hearts perfused with serum from animals treated with TNF 18-22 hours earlier, exhibited significant impairment and decreased rate of relaxation compared to controls (DeMeules et al, 1992). Similar myocardial depressing effects may possibly be inflicted by continuous exposure of the heart to TNF, circulating in heart failure patients (Levine et al, 1990). Neutralization of the cytokine with sTNF-Rs may be useful in managing heart failure.
Heparin has been reported to bind TNF (Lantz et al, 1991). However, the significance of this observation was never examined. The effects of heparin seem to be the exact opposite of the effects of TNF, as shown by Table I in Lantz et al.
The present invention provides for the use of heparin, and/or a derivative thereof, in the preparation of a pharmaceutical composition for inhibiting the bioactivity of TNF.
The present invention also provides pharmaceutical compositions for inhibiting the bioactivity of TNF.
The invention provides further a kit for the simultaneous or sequential administration of such a composition, comprising the active ingredients together with a pharmaceutically acceptable carrier, and instructions for use.
Heparin and low molecular weight heparins have been found to inhibit the cytokine bioactivity of TNF, particularly when acting with another TNF binding protein. Heparin is a natural TNF binding protein, and probably cross-links TNF to its p55 TNF and p75 TNF-receptors. This inhibits the cytokine bioactivity of TNF by presumably interfering with trimerization of the TNF receptors. The inventors raise the above theory of action without being bound thereby. Thus, by administering heparin or a derivative thereof along with a soluble TNF receptor, the bioactivity of TNF is inhibited, and the disorders caused by excess TNF can be successfully treated. The heparin or derivative thereof can be administered simultaneously with the TNF receptor, either in separate compositions or in compositions containing both heparin or a derivative thereof and at least one soluble TNF receptor.